Human Alpha-synuclein (SNCA) ELISA Kit
- SKU:
- HUEB0256
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P37840
- Range:
- 15.6-1000 pg/mL
- ELISA Type:
- Sandwich
- Synonyms:
- SNCA, NACP, PARK1
- Reactivity:
- Human
Description
| Product Name: | Human Alpha-synuclein (SNCA) ELISA Kit |
| Product Code: | HUEB0256 |
| Alias: | Alpha-synuclein, Non-A beta component of AD amyloid, Non-A4 component of amyloid precursor, NACP, SNCA, NACP, PARK1 |
| Uniprot: | P37840 |
| Reactivity: | Human |
| Range: | 15.6-1000 pg/mL |
| Detection Method: | Sandwich |
| Size: | 96 Assay |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | SNCA: a member of the synuclein family. Abundantly expressed in the brain. Inhibits phospholipase D2 selectively. May integrate presynaptic signaling and membrane trafficking. Implicated in the pathogenesis of Parkinson's disease. A major component of amyloid plaques in the brains of patients with Alzheimer's disease. Two alternatively spliced isoforms transcripts have been identified. |
| UniProt Protein Details: | Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 4q21 Cellular Component: Golgi apparatus; nuclear outer membrane; mitochondrion; rough endoplasmic reticulum; lysosome; extracellular region; fibril; terminal button; inclusion body; cell cortex; mitochondrial respiratory chain complex I; cytosol; actin cytoskeleton; synaptic vesicle; platelet alpha granule membrane; growth cone; perinuclear region of cytoplasm; axon; cytoplasm; plasma membrane; ribosome; cell junction; nucleus Molecular Function:protein domain specific binding; identical protein binding; histone binding; zinc ion binding; kinesin binding; ferrous iron binding; microtubule binding; caspase inhibitor activity; beta-tubulin binding; magnesium ion binding; phosphoprotein binding; protein N-terminus binding; oxidoreductase activity; Hsp70 protein binding; calcium ion binding; dynein binding; protein binding; copper ion binding; phospholipase binding; phospholipid binding; tau protein binding; fatty acid binding; alpha-tubulin binding Biological Process: regulation of long-term neuronal synaptic plasticity; negative regulation of serotonin uptake; regulation of acyl-CoA biosynthetic process; adult locomotory behavior; positive regulation of apoptosis; negative regulation of norepinephrine uptake; mitochondrial membrane organization and biogenesis; microglial cell activation; response to lipopolysaccharide; positive regulation of endocytosis; dopamine biosynthetic process; negative regulation of transcription from RNA polymerase II promoter; negative regulation of caspase activity; negative regulation of monooxygenase activity; fatty acid metabolic process; positive regulation of neurotransmitter secretion; regulation of dopamine secretion; negative regulation of dopamine uptake; negative regulation of histone acetylation; calcium ion homeostasis; negative regulation of exocytosis; response to magnesium ion; negative regulation of protein amino acid phosphorylation; behavioral response to cocaine; receptor internalization; phospholipid metabolic process; fibril organization and biogenesis; synapse organization and biogenesis; dopamine uptake; negative regulation of neuron apoptosis; response to iron(II) ion; positive regulation of receptor recycling; aging; caspase activation; response to drug; neutral lipid metabolic process; protein destabilization; regulation of macrophage activation; regulation of glutamate secretion; negative regulation of microtubule polymerization; positive regulation of peptidyl-serine phosphorylation; negative regulation of dopamine metabolic process; organelle ATP synthesis coupled electron transport; regulation of locomotion; synaptic vesicle endocytosis; positive regulation of release of sequestered calcium ion into cytosol; regulation of excitatory postsynaptic membrane potential; negative regulation of transporter activity; negative regulation of apoptosis Disease: Parkinson Disease 4, Autosomal Dominant; Dementia, Lewy Body; Parkinson Disease 1, Autosomal Dominant |
| NCBI Summary: | Alpha-synuclein is a member of the synuclein family, which also includes beta- and gamma-synuclein. Synucleins are abundantly expressed in the brain and alpha- and beta-synuclein inhibit phospholipase D2 selectively. SNCA may serve to integrate presynaptic signaling and membrane trafficking. Defects in SNCA have been implicated in the pathogenesis of Parkinson disease. SNCA peptides are a major component of amyloid plaques in the brains of patients with Alzheimer's disease. Four alternatively spliced transcripts encoding two different isoforms have been identified for this gene. [provided by RefSeq, Mar 2009] |
| UniProt Code: | P37840 |
| NCBI GenInfo Identifier: | 586067 |
| NCBI Gene ID: | 6622 |
| NCBI Accession: | P37840.1 |
| UniProt Secondary Accession: | P37840,Q13701, Q4JHI3, Q6IAU6, A8K2A4, |
| UniProt Related Accession: | P37840 |
| Molecular Weight: | 140 |
| NCBI Full Name: | Alpha-synuclein |
| NCBI Synonym Full Names: | synuclein, alpha (non A4 component of amyloid precursor) |
| NCBI Official Symbol: | SNCA |
| NCBI Official Synonym Symbols: | PD1; NACP; PARK1; PARK4 |
| NCBI Protein Information: | alpha-synuclein; synuclein alpha-140; non A-beta component of AD amyloid |
| UniProt Protein Name: | Alpha-synuclein |
| UniProt Synonym Protein Names: | Non-A beta component of AD amyloid; Non-A4 component of amyloid precursor; NACP |
| Protein Family: | Alpha-synuclein |
| UniProt Gene Name: | SNCA |
| UniProt Entry Name: | SYUA_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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